KR101165860B1 - Apparatus and methods for detecting nucleic acid in biological samples - Google Patents

Abstract

The present invention discloses electric field-assisted acceleration devices and methods for biological treatment involving charged individuals, in particular including detection of target DNA in biological samples. The reaction cell has a dielectric surface and generates a field by inducing charge-separation in the dielectric material by applying a potential to an electrode in contact with the dielectric material.

Description

Apparatus and method for detecting nucleic acids in biological samples {APPARATUS AND METHODS FOR DETECTING NUCLEIC ACID IN BIOLOGICAL SAMPLES}

The present invention relates to apparatus and methods for detecting nucleic acids in biological samples. In particular, the present invention relates to novel devices and methods for detecting DNA sequences using field-assisted nucleic acid hybridization, and methods for optimizing the performance of such devices, the present invention also being charged It also extends to using electric field-assisted hybridization for any biological treatment involving entities.

The advent of high density polynucleotide (eg DNA or RNA) array techniques has transformed the basic concepts of genomic and proteomic analysis. It is an industrial aspect to convert "dot blots" into "arrays on glass slides" and then into DNA microarrays (also known as DNA chips), using large scale clinical diagnostic tests and screening treatments in practical applications. It was a big change. As well-known, a typical microarray with reaction sites in a predetermined batch on a substrate may be used for a sample with a target nucleic acid fragment having a base sequence complementary to the base sequence of a capture fragment attached to the reaction site. When exposed it will show a bonding pattern. Coupling patterns and coupling efficiencies can be detected by optical or electronic methods (eg, fluorescence labeling, current detection, or impedance measurements can be included) when appropriate detection mechanisms are used.

The use of electrically-assisted nucleic acid hybridization is a known technique for the analysis of biological samples containing DNA such as blood, plasma, urine and the like. Typically, chips for DNA detection are formed from one of a variety of materials including glass, silica and metals. On the surface of the chip, many electrical contacts are formed using known techniques. To detect specific DNA sequences in a biological sample, a capture probe consisting of complementary DNA fragments is attached to the chip surface by an adhesion layer, typically an agarose gel. If the biological sample contains the target DNA, the target DNA will bind to the complementary DNA fragment by hybridization, and various imaging techniques can be used to detect this hybridization and thus the presence of the target DNA in the sample.

Conventional technology

By using electrodes, the nucleic acid fragments can be electrically charged and thus attracted to specific sites by electrostatic attraction. In addition, by applying an appropriate current, the hybridization process can be accelerated, and thus the detection process is also accelerated. However, it is not possible to use the electrodes in direct contact with nucleic acid fragments due to the risk of electrochemical degradation or electrolysis of the sample. Thus, the permeation layer / adhesion layer is generally coated on the electrode as typically shown in US Pat. Nos. 5,605,662 and 6,306,348. The permeation layer / adhesion layer is generally made of a porous material such as sol-gel material, porous hydrogel material, porous oxide, allowing selective diffusion of small ions and acting as an attachment surface to the capture probe. The pore size of the porous material is generally too small to allow larger nucleic acid fragments to pass through, thereby reducing the direct contact of the nucleic acid fragments with the electrodes.

When a voltage is applied to the electrode under the permeation layer / adhesion layer, the prior art device can provide an electrophoretic transport effect without electrochemical decomposition of the sample, and thus hybridization can be enhanced. However, it is not without drawbacks to the prior art technique that enables electrically-induced hybridization. For example, porous materials such as hydrogels and polymers are susceptible to degradation in contact with aqueous solutions, various chemicals, and numerous peripheral elements. The manufacture of sol-gel materials is expensive and complex, increasing the manufacturing cost. In addition, the porous material is brittle in nature and adsorbs and traps undesirable foreign matter such as wet hydrocarbons in the air, thereby shortening the shelf life of the device.

Summary of the Invention

In accordance with the present invention, a target nucleic acid is detected in a sample, the substrate comprising a substrate formed of one or more reaction cells comprising an attachment surface formed of a dielectric material for attachment of a nucleic acid capture probe and having a metal electrode in intimate contact with the dielectric material. An apparatus is provided. The sample may comprise a biological material and the sample may be wastewater, solution or reagent. In addition, the sample may be a biological sample such as blood, plasma or urine.

Preferably, the electrode is provided in contact with the dielectric material face directly below the attachment surface, ie opposite the attachment surface. However, it can be applied in contact with the dielectric material face or even in contact with the attachment surface itself.

In a preferred embodiment of the invention, the dielectric material may preferably be selected from the group consisting of oxides such as Al ₂ O ₃ , SiO ₂ and Ta ₂ O ₅ . For example, the metal electrode may be formed of aluminum.

In a preferred embodiment of the invention, the device comprises a first base layer, a second insulating layer formed on the base layer, and a patterned conductive region formed on the insulating layer and defining one or more metal electrodes. And a multi-layer structure comprising a third layer, and a fourth layer comprising one or more dielectric material regions, wherein each of the metal electrodes in the third layer is to be covered by a dielectric material region in the fourth layer. Can be. Preferably, the patterned conductive zones of the third layer are separated by zones formed of dielectric material. Even more preferably, the dielectric material zone in the fourth layer may be separated by a passivation material zone, which may extend to the edge of the dielectric material zone to define the reaction cell.

In another broad aspect, the present invention provides a reaction cell having an attachment surface formed of a dielectric material, providing a metal electrode in direct contact with the dielectric material, attaching a nucleic acid capture probe to the attachment surface. A method of performing field-assisted hybridization in the detection of a nucleic acid target from a biological sample, comprising the steps of: adding a sample to the reaction cell, and applying a potential to the electrode. The sample may comprise a biological material and the sample may be wastewater, solution or reagent. In addition, the sample may be a biological sample such as blood, plasma or urine.

The potential can be applied as a continuous potential or as a gentle changeable or pulsed potential.

Furthermore, in another broad aspect, the present invention provides a method of dragging an electrically-charged individual to or from a surface of a reaction cell when performing a biological reaction, the method comprising providing a dielectric material as the surface. And generating an electric field by inducing charge-separation in the dielectric material. The electrically charged individual may be a nucleic acid molecule.

In still another aspect, the present invention provides a method of patterning a metal electrode on an insulated substrate, depositing a dielectric material zone on the metal electrode, and forming a border around an upper surface edge of the dielectric material zone. To define an array of reaction cells, thereby forming an array of reaction cells for performing biological analysis.

Preferably, for example, the method comprises depositing a metal layer on an insulating surface, covering the metal layer in a desired pattern with a photoresist and removing residues of the metal layer by an etching process; Depositing a layer of dielectric material on a patterned metal such that the dielectric material covers the patterned metal and fills a region between the patterned electrodes, depositing a passivation layer on the layer of dielectric material, the Patterning a passivation layer with a photoresist and removing the passivation layer to expose the dielectric material covering the metal electrode to define a reaction cell.

Some embodiments of the invention will be described below as one example with reference to the accompanying drawings.

1 is a cross-sectional view of a chip in accordance with an embodiment of the invention.

Figure 2 is similar to Figure 1 but shows the chip in use.

3 is a schematic diagram showing the basic principle of a preferred embodiment of the present invention.

4A and 4B illustrate possible manufacturing process steps.

5 (a) and 5 (b) show the first test results.

6 (a) and 6 (b) show the second test results.

7 (a) to 7 (c) show the third test results.

1 shows a portion of an embodiment of the invention that includes three cells containing sample-containing buffer, but any number of cells may be provided, and generally it will be understood that they are formed in a rectangular array. .

Devices according to embodiments of the present invention are deposited and fabricated sequentially onto a silicon substrate using conventional deposition techniques. First, an insulating layer formed of SiO ₂ about 200 nm to 500 nm thick is formed on the Si substrate by any suitable technique including thermal oxidation or any suitable deposition technique such as sputtering, electron beam evaporation, etc. (FIG. 4A (a)). ). On top of the insulating layer, an aluminum layer of about 500 nm to 1000 nm thick is formed again using any conventional deposition technique (FIG. 4A (b)).

Once the aluminum layer is formed, it is patterned using a photoresist layer (FIG. 4A (c)), the unmasked regions are removed by etching (FIG. 4A (d)), and the photoresist is removed. (Figure 4A (e)). Then, to form the Al ₂ O ₃ zone between the aluminum section formed on a silicon dioxide substrate, and coating the Al ₂ O ₃ 50 to 500nm thick on the chip (Fig. 4A (f)). Then, using a plasma enhanced chemical vapor deposition or a similar technique, a passivation layer of (eg) Si ₃ N ₄ is deposited on Al ₂ O ₃ (FIG. 4A (g)). Thereafter, the passivation layer is patterned with photoresist (FIG. 4B (h)), and then the passivation layer is etched to expose the Al ₂ O ₃ region serving as the attachment surface of the reaction cell (FIG. 4B (i)). Finally, the photoresist is removed (Fig. 4B (j)).

The result of this manufacturing process is the multilayer structure of FIG. An aluminum zone is formed on an insulating layer of silicon dioxide, which is separated by Al ₂ O ₃ . The layer containing the discrete Al ₂ O ₃ zones to each other by the passivation material Si ₃ N ₄ is located on the aluminum section is formed on top of the aluminum and Al ₂ O ₃ layer, wherein the passivation material Si ₃ N ₄ is further down than this Cover the Al ₂ O ₃ zone separating the aluminum zone on the layer of. In addition, the passivation material extends to cover the edges of the Al ₂ O ₃ top to define the surface for the biological sample to be placed for analysis.

Therefore, in the example shown in FIG. 1, it will be understood that the chip is formed of three cells 1 to 3 each formed of Al ₂ O ₃ together with the lower aluminum pad. Although not shown in FIG. 1, when the aluminum zone is formed by etching, electrical contacts may also be formed so that a potential is applied to the aluminum zone.

Once the chip of Figure 1 has been fabricated, it can be used as the basis for many different biological tests and assays. In particular, each cell 1 to 3 in FIG. 1 may be equipped with a suitable capture probe as shown in FIG. 2. Depending on the tests performed, each cell may have the same capture probe or a different capture probe, wherein the capture probe has a nucleic acid fragment that is complementary to the fragment in the sample to be tested or analyzed. In the example of FIG. 2, all three cells 1 to 3 are identical and a drop of sample containing buffer is added to the cells to cover all three cells.

As will be appreciated by those skilled in the art, if the sample contains nucleic acid fragments complementary to the capture probes, they will be bound to the capture probes by hybridization treatment, which can be detected by known techniques. Since the nucleic acid fragments are electrically charged, the hybridization can be enhanced by providing an electric field that attracts the desired nucleic acid fragment toward the attachment surface and capture probe. The mechanism by which this can be done is shown in FIG. 3.

In particular, as shown in FIG. 3, if a potential is applied to the aluminum electrode below the cell, charge separation occurs in Al ₂ O ₃ because Al ₂ O ₃ is a dielectric material, and their polarities are below Al ₂ O _3. It will depend on the polarity of the voltage applied to the aluminum electrode. As shown on the left side of FIG. 3, if a positive potential is applied to the aluminum electrode, the upper surface of Al ₂ O ₃ will also have a positive potential that attracts the negatively charged fragments and repels the positively charged fragments. Conversely, if a negative potential is applied to the aluminum electrode, as shown on the right side of FIG. 3, the upper surface of Al ₂ O ₃ will also have a negative potential that attracts the positively charged fragments and repels the negatively charged fragments. Thus, a potential is selectively applied to an aluminum electrode that is in direct contact with the Al ₂ O ₃ attachment surface and directly underneath it to allow selective attraction / repulsion of the nucleic acid fragment, thus allowing for electrically-induced hybridization. It is to be understood that the potential can be applied in a number of different ways. For example, the potential can be a constant continuous potential and can be a gentle change or a regular pulse or any desired pattern of pulses.

A particular advantage of the present invention, at least in its preferred form, is that, in contrast to the prior art, since there is no electron transfer between the sample solution and the surface of the dielectric layer, undesirable electrochemical reactions and / or electrolysis can be completely prevented. Can be. Thus, nucleic acid fragments can be electrically attracted to the attachment surface without electrochemical degradation. In at least preferred form, a further important advantage of the electric field-assisted hybridization method and apparatus of the present invention is that the salt concentration and pH value of the sample will not change. These parameters are important factors that affect hybridization efficiency and stability of the hybridized nucleic acid fragments. Prior art electrically-assisted hybridization techniques result in significant changes in salt concentration and pH due to the electrochemical reactions and other techniques required to compensate for this effect, such as certain buffers. A further advantage of the present invention is that no electrochemical reaction takes place, which in turn means that the solution / reagent involved in the detection process will not be disturbed. There will be no bubble formation and / or precipitation during the detection process, which is important for improving the quality of the detected signal.

In addition, while preferred embodiments of the present invention have been described in connection with accelerated hybridization upon detection of nucleic acid fragments, the present invention includes an electrically-charged individual, wherein the subject is attracted to or repulsed from the surface. It should be understood that being able to control the movement of an electrically-charged individual may be more generally applicable to any desired biological method.

5-7 show a number of experimental results using the structure of FIGS. 1-3 with or without potential applied to an aluminum electrode below the cell. It is to be understood that in all of the above examples the reaction time, applied voltage and other parameters are purely exemplary and can be changed as necessary.

5A and 5B show a control in which no potential is applied to the aluminum electrode, and therefore hybridization proceeds without electrical assistance. In this example, the target oligomer in the sample is synthetic β-actin (91 bases, pure) and the hybridization time is 90 minutes. The target oligomer is present only in the sample of FIG. 5 (a) and not in the sample of FIG. 5 (b). In both FIG. 5 (a) or 5 (b), no potential is applied to the aluminum electrode, but in FIG. 5 (a) than in FIG. 5 (b) due to the presence of the target oligomer in the sample of FIG. The cell is obviously darker.

The condition in FIGS. 6 (a) and 6 (b) is that in FIG. 6 (a), the same target oligomer is provided, and in FIG. 6 (b) no target oligomer is provided. And (b) of FIG. 5. In this example, however, a potential of +10 V is applied to the aluminum electrode and the hybridization time is reduced to 10 minutes. Comparing Fig. 5 (a) with Fig. 6 (a), it is evident that the cell in Fig. 6 (a) is much darker despite the fact that the hybridization time has been substantially reduced, which is due to the applied voltage at the time of hybridization acceleration. It proves its effectiveness. The similarity of Figs. 5 (b) and 6 (b) without the target oligomer showing that no false positive results are caused by the applied voltage.

7 (a)-(c) show a third example where the target oligomer in the sample is an avian influenza virus (AIV) H5 subtype (250 bases mixed with other nonspecific oligomers). In Figures 7 (a)-(c), the hybridization time in all three cases is 10 minutes. The differences between FIGS. 7 (a)-(c) are as follows: In FIG. 7 (a), a target oligomer is present in the sample, and a potential of + 10V is applied to the aluminum electrode under the cell; In Figure 7 (b) no target oligomer is present in the sample, and a potential of + 10V is applied to the aluminum electrode under the cell; In FIG. 7C, the target oligomer is present in the sample, but no potential is applied to the aluminum electrode under the cell. This example shows once again that only 10 minutes of hybridization time and applying a + 10V potential to the electrode accelerates hybridization and generates a strong signal (very dark region in the cell of FIG. 7 (a)). By comparison, the similarity of appearance between Figure 7 (b) (no target and potential applied) and Figure 7 (c) (target but no potential applied) is the same time (10 minutes) without electrically assisted hybridization. Shows that it is impossible to achieve effective hybridization within.

At least in its applied form, the present invention can be applied to a large number of possible uses, and allows for electric field-assisted hybridization and / or other biological treatment of nucleic acids at high performance and at a faster rate, and provides a simple low cost device. The present invention uses the principle of charge-separation in dielectric materials in contact with the electrode to which a potential is applied. In the embodiments described above, the electrode is aluminum and the dielectric material is Al ₂ O _3, but other combinations of metal electrodes and dielectric attachment surfaces are also possible. For example, SiO ₂ , Ta ₂ O ₅ can be used as the oxide based dielectric material for the attachment surface.

In contrast to prior art devices using penetrating layers, the oxide based dielectric layer in direct contact with the electrode is robust, dense and chemically inert to most acids, alkalis and other reagents commonly used in biological reactions. Provide structure. In addition, the structure is stable to peripheral elements such as temperature and humidity and can be less subject to physical damage. The device of the present invention has lower manufacturing costs and can be manufactured very easily using standard deposition techniques and other microelectronic manufacturing techniques. Indeed, the use of such microelectronic deposition and fabrication techniques in the manufacture of the device of the present invention has the advantage that the device can be easily incorporated into other devices fabricated using the same or similar techniques.

Claims (34)

An apparatus for detecting a target nucleic acid in a sample, comprising a substrate formed from one or more reaction cells, the reaction cell comprising a layer formed of a dielectric material defining an attachment surface for attachment of a nucleic acid capture probe, the metal An electrode is provided to make direct contact with the layer formed of the dielectric material, and the layer formed of the dielectric material generates a flow of current between the layer formed of the dielectric material and the sample in the reaction cell when a potential is applied to the metal electrode. Or is structured in such a way that charge separation occurs in the layer formed of the dielectric material without electron transfer. The method of claim 1, And the electrode is in contact with a surface opposite the attachment surface of the dielectric material. The method of claim 1, And the dielectric material is an oxide. The method of claim 3, wherein Wherein said dielectric material is selected from the group consisting of Al ₂ O ₃ , SiO _2, and Ta ₂ O ₅ . The method of claim 1, And the electrode is formed of aluminum. The method of claim 1, Wherein said dielectric material comprises Al ₂ O ₃ and said electrode is formed of aluminum. The method of claim 1, A third layer comprising a first base layer, a second insulating layer formed on the first base layer, a patterned conductive region formed on the second insulating layer and defining one or more metal electrodes; and And a multi-layer structure comprising a fourth layer comprising at least one dielectric material zone, wherein each of the metal electrodes in the third layer is covered by a dielectric material zone in the fourth layer. The method of claim 7, wherein Wherein the patterned conductive region of the third layer is separated by a region formed of a dielectric material. The method of claim 7, wherein Wherein the dielectric material zone of the fourth layer is separated by a passivation material zone. The method of claim 9, The passivation material zone extends to an edge of the dielectric material zone to define the reaction cell. The method of claim 1, And the sample comprises a biological material. An apparatus for detecting a target biological material in a sample, comprising a substrate formed of one or more reaction cells, the reaction cell comprising a surface formed of a dielectric material, the metal electrode being provided in direct contact with the dielectric material, the dielectric The material is structured in such a way that applying a potential to the metal electrode causes charge separation within the dielectric material without the flow of current or electron transfer between the electrode and the sample in contact with the dielectric material. Wherein the device detects a target biological material in a sample. Providing a reaction cell having a layer formed of a dielectric material defining an attachment surface, providing a metal electrode in direct contact with the layer formed of the dielectric material, attaching a nucleic acid capture probe to the layer formed of the dielectric material Adding a sample to the reaction cell, and applying a potential to the metal electrode to form a layer of the dielectric material and a layer formed of the dielectric material with no current flow or electron transfer between the sample. A method for performing field-assisted hybridization in the detection of nucleic acid targets from a sample, comprising causing charge separation to occur. The method of claim 13, And the electrode is provided to contact the surface of the layer formed of the dielectric material opposite the attachment surface. The method of claim 13, Wherein the potential is a potential to be applied continuously. The method of claim 13, Wherein said potential is a potential applied as a series pulse. The method of claim 13, And the sample comprises a biological material. In carrying out a biological reaction, a method of attracting or repulsing an electrically charged entity to the surface of a reaction cell, Providing a layer formed of a dielectric material defining the surface and inducing charge separation in the layer formed of the dielectric material without generating a flow of current or transfer of electrons between the layer and the sample formed with the dielectric material Thereby generating an electric field. The method of claim 18, Disposing the electrode in direct contact with the layer formed of the dielectric material and applying a potential to the electrode to induce charge separation in the layer formed of the dielectric material. 20. The method of claim 19, A continuous potential is applied to the electrode. 20. The method of claim 19, Applying a pulsed potential to the electrode. 20. The method of claim 19, And the electrode is arranged to contact the surface of the dielectric material opposite the surface from which the charged object is attracted or repels. A method of accelerating the detection process of biological molecules carried out in a reaction cell, Providing a layer formed of a dielectric material defining an attachment surface of the reaction cell and without generating a flow of current or movement of electrons between the layer formed of the dielectric material and a biological molecule or medium to which the biological molecule belongs Generating an electric field by inducing charge separation in the dielectric material. 24. The method of claim 23, The molecule is a nucleic acid molecule. A method of forming an array of reaction cells for performing a biological analysis, the method comprising: patterning a metal electrode on an insulating substrate, depositing a region of dielectric material on the metal electrode, and the dielectric Delimiting the reaction cell by forming a border around an upper surface edge of the material zone, wherein the dielectric material causes a flow of current between the electrode and a sample in the reaction cell when a potential is applied to the metal electrode; A method of forming an array of reaction cells for performing biological analysis, wherein the deposition is carried out in such a way that charge separation occurs in the dielectric material without transfer of electrons. 26. The method of claim 25, Depositing a metal layer on an insulating surface, covering the metal layer of a desired pattern with photoresist and removing the remaining portion of the metal layer by an etching process to form a patterned metal, the patterned Depositing a layer of dielectric material on a metal such that the dielectric material covers the patterned metal and fills a region between the patterned metal, depositing a passivation layer on the dielectric material layer, the passivation Patterning the layer with a photoresist, and removing the passivation layer to expose the dielectric material covering the metal electrode to define the reaction cell. The method of claim 13, And the electrode is in contact with the side of the layer formed of the dielectric material opposite the attachment surface. The method of claim 13, The dielectric material is an oxide. 29. The method of claim 28, The dielectric material is selected from the group consisting of Al ₂ O ₃ , SiO ₂ and Ta ₂ O ₅ . The method of claim 13, And the electrode is formed of aluminum. The method of claim 13, Wherein said dielectric material comprises Al ₂ O ₃ and said electrode is formed of aluminum. The method of claim 13, Wherein the layer formed of the dielectric material is impermeable to molecules in the sample. The method of claim 18, Wherein the layer formed of the dielectric material is impermeable to components in the sample containing the electrically charged individual. 24. The method of claim 23, Wherein the layer formed of the dielectric material is impermeable and nonporous with respect to components in the sample containing the biological molecule.

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